Protease is an enzyme that cleaves a specific peptide bond of proteins. In biological organisms, proteases having specific proteolytic activities, and their inhibitors are involved in regulation of various biological functions. In diverse biological processes, biologically necessary functions can be activated and regulated by proteolytic cleavage of a polyprotein precursor by a protease that results in formation of active proteins. Examples include blood coagulation, immuno-defensive processes, selective transports of proteins through intracellular membranes, viral proliferation in a host cell, etc. Therefore, protease is a major target in the development of specific protease inhibitors as new drugs.
Viral protease inhibitor is a representative example of a protease inhibitor developed as a new drug. Since the viral protease participates in the activation of polyprotein precursors via proteolytic cleavage, the protease is an essential element for the initiation of the viral proliferation and thus for the correct capsid assembly of replicated viruses in the host cell.
Protease inhibitors have been developed to block the proliferation of HIV that causes the acquired immune deficiency syndrome (AIDS). For example, amprenavir, nelfinavir, indinavir, ritonavir, and squinavir have been approved by FDA as drugs for inhibiting the HIV protease, and lopinavir and efavirenz are under clinical studies. Patients who were administered those medicines showed that the number of HIV particulates decreased to about 10% of that before the medicinal treatment. This shows that the protease inhibitor can be used as an efficient medicine. However, several side effects were reported during such treatments (Miller, T. L. et al., 2001), and mutants having mutated protease genes were reported in the cases of prolonged treatments (Jacobsen, H. et al., 1996; Cote, H. et al., 2001). Therefore, more diverse protease inhibitors that can specifically block the proliferation of various mutant HIV viruses need to be developed.
Protease inhibitors have been studied to inhibit other human and animal viruses such as HCV (Kasai, N. et al., 2001) and HERV (Kuhelj, R. et al., 2001). Researches for plant virus diseases have been also performed based on the same concept. For example, inhibition of the proteolytic cleavage of the polyproteins produced by TEV (tobacco etch virus) and PVY (potato virus Y) has been studied by expressing a recombinant protein as a protease inhibitor in a transgenic plant (Gutierres-Campos, R. et al., 1999). A study to identify proteolytic sites of a protease from a plant virus has been also performed (Yoon, H. Y. et al., 2000).
There have been attempts to develop protease inhibitors. For example, in many cases, screening of protease inhibitors has been performed by measuring cleavage of a substrate using electrophoresis, after a protease, its substrate peptide or protein, and a candidate chemical were mixed to react in vitro. As the importance of the proteolytic site has been recognized, peptides having amino acid sequences that are similar to the proteolytic site have been synthesized and used to find protease inhibitors (Kettner, C. A. and Korant, B. D., 1987). As it becomes easier to determine tertiary structures of proteins and also possible to design chemicals using computer simulation, many researches have been conducted to design and synthesize molecules that specifically bind to the active site of the enzyme (Wlodawer, A. and Erickson, J. W., 1993; Rodgers, J. D. et al., 1998; Mardis, K. L. et al., 2001). Also, attempts were made to use fluorescence-labeled substrates in order to increase the efficiency of the protease activity measurement (Ermolief, J. et al., 2000), and to use fragments of antibody expressed in the periplasm of E. Coli as protease inhibitors in order to enlarge the skeletal structure of the protease inhibitor (Kasai, N. et al., 2001).
Most of the protease activity screening methods used currently are performed in vitro. However, in the in vitro screening method, it is not possible to examine various complicated effects such as the transport efficiency of drug candidates into the cell, the stability and cytotoxicity of drug candidates in the cell, etc. Many additional time-consuming experiments are thus necessary before examining the drug candidates selected by the in vitro screening method in a living body. Therefore, a simpler and more generalized in vivo screening method needs to be developed to examine the cellular functions of the protease inhibitor candidates and also to screen more specific protease inhibitors.
There have been efforts to detect proteolytic cleavage and screening of protease inhibitors in more in vivo or in vivo-like conditions.
For example, there has been a report of a method using protease present in isolated vesicles (Hook, V. Y., 2001). Other disclosed methods include in situ zymography using a tissue section (Yi, C.-F. et al., 2001), and a method of treating cells with a polypeptide substrate of a protease (Kuhelj, R. et al., 2001).
However there is increasing recognition that these and related methods are associated with shortcomings.
For example, many of the methods are believed to only approximate in vivo environments. Accordingly, such methods do not always reflect intracellular environments that may substantially impact protease function.
Moreover, many of the prior methods are believed to be limited in terms of sensitivity, selectivity, and convenience. These and other drawbacks are believed to have lowered the efficiency and reliability of past screening attempts.
It would be desirable to have better in vivo methods for detecting protease inside cells that are more sensitive and easier to use. It would be especially desirable to have in vivo methods that can be readily adapted to detect inhibitors of mammalian and viral proteases.